Detecting insulitis in type 1 diabetes with ultrasound phase-change contrast agents.

Type 1 diabetes (T1D) results from immune infiltration and destruction of insulin-producing ß cells within the pancreatic islets of Langerhans (insulitis). Early diagnosis during presymptomatic T1D would allow for therapeutic intervention prior to substantial ß-cell loss at onset. There are limited methods to track the progression of insulitis and ß-cell mass decline. During insulitis, the islet microvasculature increases permeability, such that submicron-sized particles can extravasate and accumulate within the islet microenvironment. Ultrasound is a widely deployable and cost-effective clinical imaging modality. However, conventional microbubble contrast agents are restricted to the vasculature. Submicron nanodroplet (ND) phase-change agents can be vaporized into micron-sized bubbles, serving as a microbubble precursor. We tested whether NDs extravasate into the immune-infiltrated islet microenvironment. We performed ultrasound contrast-imaging following ND infusion in nonobese diabetic (NOD) mice and NOD;Rag1ko controls and tracked diabetes development. We measured the biodistribution of fluorescently labeled NDs, with histological analysis of insulitis. Ultrasound contrast signal was elevated in the pancreas of 10-wk-old NOD mice following ND infusion and vaporization but was absent in both the noninfiltrated kidney of NOD mice and the pancreas of Rag1ko controls. High-contrast elevation also correlated with rapid diabetes onset. Elevated contrast was also observed as early as 4 wk, prior to mouse insulin autoantibody detection. In the pancreata of NOD mice, infiltrated islets and nearby exocrine tissue were selectively labeled with fluorescent NDs. Thus, contrast ultrasound imaging with ND phase-change agents can detect insulitis prior to diabetes onset. This will be important for monitoring disease progression, to guide and assess preventative therapeutic interventions for T1D.

Photoacoustic Vaporization of Endoskeletal Droplets Loaded with Zinc Naphthalocyanine.

Vaporizable endoskeletal droplets are solid hydrocarbons in liquid fluorocarbon droplets in which melting of the hydrocarbon phase leads to the vaporization of the fluorocarbon phase. In prior work, vaporization of the endoskeletal droplets was achieved thermally by heating the surrounding aqueous medium. In this work, we introduce a near-infrared (NIR) optically absorbing naphthalocyanine dye (zinc 2,11,20,29-tetra-tert-butyl-2,3-naphthalocynanine) into the solid hydrocarbon (eicosane, n-C20H42) core of liquid fluorocarbon (C5F12) drops suspended in an aqueous medium. Droplets with a uniform diameter of 11.7 ± 0.7 µm were formed using a flow-focusing microfluidic device. The solid hydrocarbon formed a crumpled spherical structure within the liquid fluorocarbon droplet. The photoactivation behavior of these dye-containing endoskeletal droplets was investigated using NIR laser irradiation. When exposed to a pulsed laser of 720 nm wavelength, the dye-containing droplets vaporized at an average laser fluence of 65 mJ/cm2, whereas blank droplets without the dye did not vaporize at any fluence up to 100 mJ/cm2. Furthermore, dye-loaded droplets with a smaller, polydisperse size distribution were prepared using a simple shaking method and studied in a flow phantom for their photoacoustic signal and ultrasound contrast imaging. These results demonstrate that dye-containing endoskeletal droplets can be made to vaporize by externally applied optical energy. Such droplets may be useful for a variety of photoacoustic applications for sensing, imaging, and therapy.

Nanobubbles are Non-Echogenic for Fundamental-Mode Contrast-Enhanced Ultrasound Imaging.

Microbubbles (1-10 µm diameter) have been used as conventional ultrasound contrast agents (UCAs) for applications in contrast-enhanced ultrasound (CEUS) imaging. Nanobubbles (<1 µm diameter) have recently been proposed as potential extravascular UCAs that can extravasate from the leaky vasculature of tumors or sites of inflammation. However, the echogenicity of nanobubbles for CEUS remains controversial owing to prior studies that have shown very low ultrasound backscatter. We hypothesize that microbubble contamination in nanobubble formulations may explain the discrepancy. To test our hypothesis, we examined the size distributions of lipid-coated nanobubble and microbubble suspensions using multiple sizing techniques, examined their echogenicity in an agar phantom with fundamental-mode CEUS at 7 MHz and 330 kPa peak negative pressure, and interpreted our results with simulations of the modified Rayleigh-Plesset model. We found that nanobubble formulations contained a small contamination of microbubbles. Once the contribution from these microbubbles is removed from the acoustic backscatter, the acoustic contrast of the nanobubbles was shown to be near noise levels. This result indicates that nanobubbles have limited utility as UCAs for CEUS.

Effect of Thermal History and Hydrocarbon Core Size on Perfluorocarbon Endoskeletal Droplet Vaporization.

Vaporizable hydrocarbon-in-fluorocarbon endoskeletal droplets are a unique category of phase-change emulsions with interesting physical and thermodynamic features. Here, we show microfluidic fabrication of various morphologies, such as solid-in-liquid, liquid-in-solid, and Janus-type, of complex solid n-C20H42 or n-C21H44 and liquid n-C5F12 droplets. Furthermore, we investigated the vaporization behavior of these endoskeletal droplets, focusing on the effects of heat treatment and core size. Comparison of vaporization and differential scanning calorimetry results indicated that vaporization occurs prior to melting of the bulk hydrocarbon phase for C20H42/C5F10 droplets and near the rotator phase for C21H44/C5F10 droplets. We found that heat treatment of the droplets increased the fraction of droplets that vaporized and also increased the vaporization temperature of the droplets, although the effect was temporary. Furthermore, we found that changing the relative size of the solid hydrocarbon core compared to the surrounding liquid shell increased the vaporization temperature and the vaporizing fraction. Taken together, these data support the hypothesis that surface melting behavior exhibited by the linear alkane may trigger the fluorocarbon vaporization event. These results may aid in the understanding of the interfacial thermodynamics and transport and the engineering of novel vaporizable endoskeletal droplets for biomedical imaging and other applications.

Contrast-Enhanced Sonography with Biomimetic Lung Surfactant Nanodrops.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilize an expanding gas surface by employing the lung surfactant replacement, beractant. Lung surfactant contains a suite of lipids and proteins that provide efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and resolidification. We then harnessed this unique functionality to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

Acoustic Nanodrops for Biomedical Applications.

Acoustic nanodrops are designed to vaporize into ultrasound-responsive microbubbles, which presents certain challenges nonexistent for conventional nano-emulsions. The requirements of biocompatibility, vaporizability and colloidal stability has focused research on perfluorocarbons (PFCs). Shorter PFCs yield better vaporizability via their lower critical temperature, but they also dissolve more easily owing to their higher vapor pressure and solubility. Thus, acoustic nanodrops have required a tradeoff between vaporizability and colloidal stability in vivo. The recent advent of vaporizable endoskeletal droplets, which are both stable and vaporizable, may have solved this problem. The purpose of this review is to justify this premise by pointing out the beneficial properties of acoustic nanodrops, providing an analysis of vaporization and dissolution mechanisms, and reviewing current biomedical applications.

Bubble Inflation Using Phase-Change Perfluorocarbon Nanodroplets as a Strategy for Enhanced Ultrasound Imaging and Therapy.

Phase-change perfluorocarbon microdroplets were introduced over 2 decades ago to occlude downstream vessels in vivo. Interest in perfluorocarbon nanodroplets has recently increased to enable extravascular targeting, to rescue the weak ultrasound signal of perfluorocarbon droplets by converting them to microbubbles and to improve ultrasound-based therapy. Despite great scientific interest and advances, applications of phase-change perfluorocarbon agents have not reached clinical testing because of efficacy and safety concerns, some of which remain unexplained. Here, we report that the coexistence of perfluorocarbon droplets and microbubbles in blood, which is inevitable when droplets spontaneously or intentionally vaporize to form microbubbles, is a major contributor to the observed side effects. We develop the theory to explain why the coexistence of droplets and microbubbles results in microbubble inflation induced by perfluorocarbon transfer from droplets to adjacent microbubbles. We also present the experimental data showing up to 6 orders of magnitude microbubble volume expansion, which occludes a 200 µm tubing in the presence of perfluorocarbon nanodroplets. More importantly, we demonstrate that the rate of microbubble inflation and ultimate size can be controlled by manipulating formulation parameters to tailor the agent's design for the potential theranostic application while minimizing the risk to benefit ratio.

Click Conjugation of Cloaked Peptide Ligands to Microbubbles.

Interest in the use of targeted microbubbles for ultrasound molecular imaging (USMI) has been growing in recent years as a safe and efficacious means of diagnosing tumor angiogenesis and assessing response to therapy. Of particular interest are cloaked microbubbles, which improve specificity by concealing the ligand from blood components until they reach the target vasculature, where the ligand can be transiently revealed for firm receptor-binding by ultrasound acoustic radiation force pulses. Herein, a bio-orthogonal "click" conjugation chemistry is introduced to decorate the surface of cloaked 4-5-µm-diameter microbubbles as part of a sterile and reproducible production process. Azido-functionalized antagonists for the angiogenic biomarkers αVß3 integrin (cRGD) and VEGFR2 (A7R) proteins were conjugated to bimodal-brush microbubbles via strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) click chemistry. Ligand conjugation was validated by epifluorescent microscopy, flow cytometry, and Fourier-transform infrared spectroscopy. Sterility was validated by bacterial culture and endotoxin analysis. Additionally, clinically normal dogs receiving escalating microbubble doses were shown to experience no pathologic changes in physical examination, complete blood count, serum biochemistry profile, or coagulation panel. This bio-orthogonal microbubble conjugation process for cloaked peptide ligands may be leveraged for future USMI studies of tumor angiogenesis for translation to preclinical and clinical applications.

Hydrostatic Pressurization of Lung Surfactant Microbubbles: Observation of a Strain-Rate Dependent Elasticity.

The microbubble offers a unique platform to study lung surfactant mechanics at physiologically relevant geometry and length scale. In this study, we compared the response of microbubbles (∼15 µm initial radius) coated with pure dipalmitoyl-phosphatidylcholine (DPPC) versus naturally derived lung surfactant (SURVANTA) when subjected to linearly increasing hydrostatic pressure at different rates (0.5-2.3 kPa/s) at room temperature. The microbubbles contained perfluorobutane gas and were submerged in buffered saline saturated with perfluorobutane at atmospheric pressure. Bright-field microscopy showed that DPPC microbubbles compressed spherically and smoothly, whereas SURVANTA microbubbles exhibited wrinkling and smoothing cycles associated with buckling and collapse. Seismograph analysis showed that the SURVANTA collapse amplitude was constant, but the collapse rate increased with the pressurization rate. An analysis of the pressure-volume curves indicated that the dilatational elasticity increased during compression for both shell types. The initial dilatational elasticity for SURVANTA was nearly twice that of DPPC at higher pressurization rates (>1.5 kPa/s), producing a pressure drop of up to 60 kPa across the film prior to condensation of the perfluorobutane core. The strain-rate dependent stiffening of SURVANTA shells likely arises from their composition and microstructure, which provide enhanced in-plane monolayer rigidity and lateral repulsion from surface-associated collapse structures. Overall, these results provide new insights into lung surfactant mechanics and collapse behavior during compression.

Single Microbubble Measurements of Lipid Monolayer Viscoelastic Properties for Small-Amplitude Oscillations.

Lipid monolayer rheology plays an important role in a variety of interfacial phenomena, the physics of biological membranes, and the dynamic response of acoustic bubbles and drops. We show here measurements of lipid monolayer elasticity and viscosity for very small strains at megahertz frequency. Individual plasmonic microbubbles of 2-6 µm radius were photothermally activated with a short laser pulse, and the subsequent nanometer-scale radial oscillations during ring-down were monitored by optical scatter. This method provided average dynamic response measurements of single microbubbles. Each microbubble was modeled as an underdamped linear oscillator to determine the damping ratio and eigenfrequency, and thus the lipid monolayer viscosity and elasticity. Our nonisothermal measurement technique revealed viscoelastic trends for different lipid shell compositions. We observed a significant increase in surface elasticity with the lipid acyl chain length for 16 to 20 carbons, and this effect was explained by an intermolecular forces model that accounts for the lipid composition, packing, and hydration. The surface viscosity was found to be equivalent for these lipid shells. We also observed an anomalous decrease in elasticity and an increase in viscosity when increasing the acyl chain length from 20 to 22 carbons. These results illustrate the use of a novel nondestructive optical technique to investigate lipid monolayer rheology in new regimes of frequency and strain, possibly elucidating the phase behavior, as well as how the dynamic response of a microbubble can be tuned by the lipid intermolecular forces.

Fluorocarbon nanodrops as acoustic temperature probes.

This work investigated the use of superheated fluorocarbon nanodrops for ultrasound thermal imaging and the use of mixed fluorocarbons for tuning thermal and acoustic thresholds for vaporization. Droplets were fabricated by condensing phospholipid-coated microbubbles containing C3F8 and C4F10 mixed at various molar ratios. Vaporization temperatures first were measured in a closed system by optical transmission following either isothermal pressure release or isobaric heating. The vaporization temperature was found to depend linearly on the percentage of C4F10 in the droplet core, indicating excellent tunability under these fluorocarbon-saturated conditions. Vaporization temperatures were then measured in an open system using contrast-enhanced ultrasound imaging, where it was found that the mixed droplets behaved like pure C4F10 drops. Additionally, the critical mechanical index for vaporization was measured at the limits of therapeutic hyperthermia (37 and 60 °C), and again the mixed droplets were found to behave like pure C4F10 drops. These results suggested that C3F8 preferentially dissolves out of the droplet core in open systems, as shown by a simple mass transfer model of multicomponent droplet dissolution. Finally, proof-of-concept was shown that pure C4F10 nanodrops can be used as an acoustic temperature probe. Overall, these results not only demonstrate the potential of superheated fluorocarbon emulsions for sonothermetry but also point to the limits of tunability for fluorocarbon mixtures owing to preferential release of the more soluble species to the atmosphere.

Thermal activation of superheated lipid-coated perfluorocarbon drops.

This study explored the thermal conditions necessary for the vaporization of superheated perfluorocarbon nanodrops. Droplets C3F8 and C4F10 coated with a homologous series of saturated diacylphosphatidylcholines were formed by condensation of 4 µm diameter microbubbles. These drops were stable at room temperature and atmospheric pressure, but they vaporized back into microbubbles at higher temperatures. The vaporization transition was measured as a function of temperature by laser light extinction. We found that C3F8 and C4F10 drops experienced 90% vaporization at 40 and 75 °C, respectively, near the theoretical superheat limits (80-90% of the critical temperature). We therefore conclude that the metastabilty of these phase-change agents arises not from the droplet Laplace pressure altering the boiling point, as previously reported, but from the metastability of the pure superheated fluid to homogeneous nucleation. The rate of C4F10 drop vaporization was quantified at temperatures ranging from 55 to 75 °C, and an apparent activation energy barrier was calculated from an Arrhenius plot. Interestingly, the activation energy increased linearly with acyl chain length from C14 to C20, indicating that lipid interchain cohesion plays an important role in suppressing the vaporization rate. The vaporized drops (microbubbles) were found to be unstable to dissolution at high temperatures, particularly for C14 and C16. However, proper choice of the fluorocarbon and lipid species provided a nanoemulsion that could undergo at least ten reversible condensation/vaporization cycles. The vaporization properties presented in this study may facilitate the engineering of tunable phase-shift particles for diagnostic imaging, targeted drug delivery, tissue ablation, and other applications.

Optically induced resonance of nanoparticle-loaded microbubbles.

We report on the optical excitation and detection of resonant microbubble oscillations. Optically absorbing nanoparticles were attached to the shell of a lipid-encapsulated microbubble, allowing for optical pulsing to photothermally drive the microbubble into resonance. A modified optical microscope was used to track the bubble wall radius as a function of time using light scattering. The microbubble response from a nanosecond laser pulse was measured, and the eigenfrequency and vibrational amplitude were determined and compared to theory. The ability to optically drive microbubble oscillations may have applications in basic studies of bubble dynamics and biomedical imaging and therapy.

Condensation phase diagrams for lipid-coated perfluorobutane microbubbles.

The goal of this study was to explore the thermodynamic conditions necessary to condense aqueous suspensions of lipid-coated gas-filled microbubbles into metastable liquid-filled nanodrops as well as the physicochemical mechanisms involved with this process. Individual perfluorobutane microbubbles and their lipid shells were observed as they were pressurized at 34.5 kPa s(-1) in a microscopic viewing chamber maintained at temperatures ranging from 5 to 75 °C. The microbubbles contracted under pressure, ultimately leading to either full dissolution or microbubble-to-nanodrop condensation. Temperature-pressure phase diagrams conveying condensation and stability transitions were constructed for microbubbles coated with saturated diacylphosphatidylcholine lipids of varying acyl chain length (C16 to C24). The onset of full dissolution was shifted to higher temperatures with the use of longer acyl chain lipids or supersaturated media. Longer chain lipid shells resisted both dissolution of the gas core and mechanical compression through a pronounced wrinkle-to-fold collapse transition. Interestingly, the lipid shell also provided a mechanical resistance to condensation, shifting the vapor-to-liquid transition to higher pressures than for bulk perfluorobutane. This result indicated that the lipid shell can provide a negative apparent surface tension under compression. Overall, the results of this study will aid in the design and formulation of vaporizable fluorocarbon nanodrops for various applications, such as diagnostic ultrasound imaging, targeted drug delivery, and thermal ablation.

Targeting diffuse midline gliomas: The promise of focused ultrasound-mediated blood-brain barrier opening.

Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma, have among the highest mortality rates of all childhood cancers, despite recent advancements in cancer therapeutics. This is partly because, unlike some CNS tumors, the blood-brain barrier (BBB) of DMG tumor vessels remains intact. The BBB prevents the permeation of many molecular therapies into the brain parenchyma, where the cancer cells reside. Focused ultrasound (FUS) with microbubbles has recently emerged as an innovative and exciting technology that non-invasively permeabilizes the BBB in a small focal region with millimeter precision. In this review, current treatment methods and biological barriers to treating DMGs are discussed. State-of-the-art FUS-mediated BBB opening is then examined, with a focus on the effects of various ultrasound parameters and the treatment of DMGs.

Effect of Poly(ethylene glycol) Configuration on Microbubble Pharmacokinetics.

Microbubbles (MBs) hold substantial promise for medical imaging and therapy; nonetheless, knowledge gaps persist between composition, structure, and in vivo performance, especially with respect to pharmacokinetics. Of particular interest is the role of the poly(ethylene glycol) (PEG) layer, which is thought to shield the MB against opsonization and rapid clearance but is also known to cause an antibody response upon multiple injections. The goal of this study was, therefore, to elucidate the role of the PEG layer in circulation persistence of MBs in the naïve animal (prior to an adaptive immune response). Here, we directly observe the number and size of individual MBs obtained from blood samples, unifying size and concentration into the microbubble volume dose (MVD) parameter. This approach enables direct evaluation of the pharmacokinetics of intact MBs, comprising both the lipid shell and gaseous core, rather than separately assessing the lipid or gas components. We examined the in vivo circulation persistence of 3 µm diameter phospholipid-coated MBs with three different mPEG2000 content: 2 mol % (mushroom), 5 mol % (intermediate), and 10 mol % (brush). MB size and concentration in the blood were evaluated by a hemocytometer analysis over 30 min following intravenous injections of 20 and 40 µL/kg MVD in Sprague-Dawley rats. Interestingly, pharmacokinetic analysis demonstrated that increasing PEG concentration on the MB surface resulted in faster clearance. This was evidenced by a 1.6-fold reduction in half-life and area under the curve (AUC) (p < 0.05) in the central compartment. Conversely, the AUC in the peripheral compartment increased with PEG density, suggesting enhanced MB trapping by the mononuclear phagocyte system. This was supported by an in vitro assay, which showed a significant rise in complement C3a activation with a higher PEG content. In conclusion, a minimal PEG concentration on the MB shell (mushroom configuration) was found to prolong circulation and mitigate immunogenicity.

In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.

In designing targeted contrast agent materials for imaging, the need to present a targeting ligand for recognition and binding by the target is counterbalanced by the need to minimize interactions with plasma components and to avoid recognition by the immune system. We have previously reported on a microbubble imaging probe for ultrasound molecular imaging that uses a buried-ligand surface architecture to minimize unwanted interactions and immunogenicity. Here we examine for the first time the utility of this approach for in vivo molecular imaging. In accordance with previous results, we showed a threefold increase in circulation persistence through the tumor of a fibrosarcoma model in comparison with controls. The buried-ligand microbubbles were then activated for targeted adhesion through the application of noninvasive ultrasound radiation forces applied specifically to the tumor region. Using a clinical ultrasound scanner, microbubbles were activated, imaged, and silenced. The results showed visually conspicuous images of tumor neovasculature and a twofold increase in ultrasound radiation force enhancement of acoustic contrast intensity for buried-ligand microbubbles, whereas no such increase was found for exposed-ligand microbubbles. We therefore conclude that the use of acoustically active buried-ligand microbubbles for ultrasound molecular imaging bridges the demand for low immunogenicity with the necessity of maintaining targeting efficacy and imaging conspicuity in vivo.

Targeted Microbubbles for Drug, Gene, and Cell Delivery in Therapy and Immunotherapy.

Microbubbles are 1-10 µm diameter gas-filled acoustically-active particles, typically stabilized by a phospholipid monolayer shell. Microbubbles can be engineered through bioconjugation of a ligand, drug and/or cell. Since their inception a few decades ago, several targeted microbubble (tMB) formulations have been developed as ultrasound imaging probes and ultrasound-responsive carriers to promote the local delivery and uptake of a wide variety of drugs, genes, and cells in different therapeutic applications. The aim of this review is to summarize the state-of-the-art of current tMB formulations and their ultrasound-targeted delivery applications. We provide an overview of different carriers used to increase drug loading capacity and different targeting strategies that can be used to enhance local delivery, potentiate therapeutic efficacy, and minimize side effects. Additionally, future directions are proposed to improve the tMB performance in diagnostic and therapeutic applications.

Monodispersity Increases Adhesion Efficiency and Specificity for Ultrasound-Targeted Microbubbles.

Ultrasound molecular imaging with targeted microbubbles (MBs) can be used to noninvasively diagnose, monitor, and study the progression of different endothelial-associated diseases. Acoustic radiation force (Frad) can initiate and enhance MB adhesion at the target site. The goal of this study was to elucidate the effects of various MB parameters on Frad targeting. Monodisperse or polydisperse MBs with the immune-stealth cloaked (buried)-ligand architecture were conjugated with targeting RGD or nonspecific isotype control RAD peptides and then pumped through an αvß3 integrin-coated microvessel phantom at a wall shear stress of 3.5 dyn/cm2. Targeting was assessed by measuring MB attachment for varying Frad time and frequency, as well as MB concentration and size distribution. We first confirmed that primary Frad is necessary to target the cloaked-ligand MBs. MB targeting increased monotonically with αvß3 integrin density and Frad time. MB attachment and, to a lesser extent specificity, also increased when driven by Frad near resonance. MB targeting increased with MB concentration, although a shift in behavior was observed with increasing MB-MB interactions and aggregations forming from secondary Frad effects as MB concentration was increased. These secondary Frad effects reduced targeting specificity. Finally, after having validated our approach by testing different parameters with the appropriate controls, we then determined the effects of monodispersity on adhesion efficiency and specific targeting. We observed that both MB targeting efficiency and specificity were greatly enhanced for monodisperse vs polydisperse MBs. Analysis of videomicroscopy images indicated that secondary Frad effects may have disproportionally inhibited targeting of polydisperse MBs. In conclusion, our in vitro results indicate that monodisperse MBs driven near resonance and at a low concentration (∼106 MB/mL) can be used to maximize the adhesion efficiency (up to 88%) and specificity of RGD-MB targeting.

Effect of Anesthetic Carrier Gas on In Vivo Circulation Times of Intravenously Administered Phospholipid Oxygen Microbubbles in Rats.

OBJECTIVE: For the treatment of tumor hypoxia, microbubbles comprising oxygen as a majority component of the gas core with a stabilizing shell may be used to deliver and release oxygen locally at the tumor site through ultrasound destruction. Previous work has revealed differences in circulation half-life in vivo for perfluorocarbon-filled microbubbles, typically used as ultrasound imaging contrast agents, as a function of anesthetic carrier gas. These differences in circulation time in vivo were likely due to gas diffusion as a function of anesthetic carrier gas, among other variables. This work has motivated studies to evaluate the effect of anesthetic carrier gas on oxygen microbubble circulation dynamics. METHODS: Circulation time for oxygen microbubbles was derived from ultrasound image intensity obtained during longitudinal kidney imaging. Studies were constructed for rats anesthetized on inhaled isoflurane with either pure oxygen or medical air as the anesthetic carrier gas. RESULTS: Results indicated that oxygen microbubbles were highly visible via contrast-specific imaging. Marked signal enhancement and duration differences were observed between animals breathing air and oxygen. Perhaps counterintuitively, oxygen microbubbles disappeared from circulation significantly faster when the animals were breathing pure oxygen compared with medical air. This may be explained by nitrogen counterdiffusion from blood into the bubble, effectively changing the gas composition of the core, as has been observed in perfluorocarbon core microbubbles. CONCLUSION: Our findings suggest that the apparent longevity and persistence of oxygen microbubbles in circulation may not be reflective of oxygen delivery when the animal is anesthetized breathing air.